Corpuscles of stannius protein, stanniocalcin

ABSTRACT

The present invention relates to human Corpuscles of Stannius polypeptides and DNA (RNA) encoding such polypeptides. Also provided is a procedure for producing such polypeptides by recombinant techniques and antagonists against such polypeptides. Also provided are methods of using the polypeptide therapeutically for treating renal, bone and heart diseases and for using the antagonists for treating hypocalcemia and osteoporosis. Also provided is a diagnostic assay to detect mutations in the nucleic acid sequence encoding the polypeptide and for altered concentrations of the polypeptide in a sample derived from a host.

This application is a continuation-in-part of a previous applicationfiled in the United States Patent and Trademark Office on Mar. 8, 1994and assigned Ser. No. 08/208,005, now U.S. Pat. No. 5,837,498.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. The polypeptide of the present invention has beenputatively identified as a human Corpuscles of Stannius protein. Theinvention also relates to inhibiting the action of such polypeptide.

Stanniocalcin (formerly known as both teleocalcin and hypocalcin) is ananti-hypercalcemic, glycoprotein hormone that is produced by theCorpuscles of Stannius, endocrine glands which are confined to bonyfishes. The polypeptide of the present invention has a high degree ofhomology at the amino acid level to the glycoprotein hormone from fishwhich is involved in the regulation of calcium levels.

The Corpuscles of Stannius protein of non-humans has been studiedextensively. Recently, a Corpuscles of Stannius protein has beenpurified and cloned from Anquilla australis. The kidneys of teleost fishhave been found to contain secretory granules, the Corpuscles ofStannius. Electron microscopy indicates that the granules are of aproteinaceous nature and may represent hormones or enzymes ofunrecognized physiological and biochemical function (Butkus, A. et al.Mol. Cell Endocrinol, 54:123-33 (1987)).

There has also been isolated and purified a glycoprotein from theCorpuscles of Stannius of trout, which is considered hypocalcin, themajor hypocalcemic hormone of fish. This product is present inrelatively large amounts in the Corpuscles of Stannius of severalspecies (i.e., European eel, tilapia goldfish, and carp). Hypocalcin istypically released from the Corpuscles of Stannius in response to anexperimentally induced increase of the blood calcium concentration.Ultrastructural observations show that after this treatment thehypocalcin-producing cell type of the corpuscles of stannius are almostcompletely degranulated. The isolated glycoprotein has an apparentmolecular weight of 54 Kda (Lafeber F. P. et al., Gen Comp. Endocrinol,69:19-30 (1988)).

Moreover, it has recently been shown that several synthetic peptidefragments of teleocalcin inhibit calcium uptake in juvenile rainbowtrout (Salmo Gairdneri). The N-terminal peptides (amino acids 1 to 20)of both eel and salmon teleocalcin significantly inhibit Ca⁴⁵ uptake atthe high point of the calcium uptake cycle (up to 75%), although theeffective doses of the peptides on a molar basis were 20 to 200 timesthat of the intact molecule. In contrast, the C-terminal fragment of eelteleocalcin (amino acids 202 to 231) did not have an inhibitory effecton calcium uptake (Milliken C. E. et al., Gen. Comp. Endocrinol,77:416-22 (1990)).

There has also been a description of the purification andcharacterization of two salmon stanniocalcins, and the examination ofthe regulation of hormone secretion in response to calcium using both anin vitro and in vivo model systems. The molecular cloning and cDNAsequence analysis of a coho salmon stanniocalcin messenger RNA (mRNA)from a salmon CS lambda gt10 cDNA library is described. Thestanniocalcin mRNA in salmon is approximately 2 Kda in length andencodes a primary translation product of 256 amino acids. The first 33residues comprise the preprotein region of the hormone, whereas theremaining 223 residues make up the mature form of the hormone (Wagner G.F. et al., Mol. Cell Endocrinol, 90:7-15 (1992)).

In accordance with one aspect of the present invention, there areprovided novel mature polypeptides of the present invention, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof. The proteins of the presentinvention are of human origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding such polypeptide,including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically activeand diagnostically or therapeutically useful fragments, analogs andderivatives thereof.

In accordance with another aspect of the present invention there areprovided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to nucleic acid sequencesencoding the polypeptides of the present invention.

In accordance with a further aspect of the present invention, there isprovided a process for producing such polypeptides by recombinanttechniques which comprises culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding apolypeptide of the present invention, under conditions promotingexpression of said protein and subsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, thereis provided processes for utilizing such polypeptides, orpolynucleotides encoding such polypeptides for therapeutic purposes, forexample, treatment of electrolyte disorders which lead to renal, boneand heart diseases, and disorders due to elevated bone resorption, e.g.osteoporosis and Paget's disease.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, in the treatmentof hypocalcemia resulting in tetany, convulsions and other relateddisorders, and osteoporosis.

In accordance with another aspect of the present invention there isprovided a method of diagnosing a disease or a susceptibility to adisease related to a mutation in the nucleic acid sequences and thepolypeptides encoded thereby of the invention.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A-1B (collectively FIG. 1) displays the cDNA sequence andcorresponding deduced amino acid sequence of the preprocessed Corpusclesof Stannius protein. The amino acid sequence is shown below using thestandard 3 letter abbreviation code.

FIG. 2 displays the comparison of the polypeptide of the presentinvention (upper line, see SEQ ID NO:2) to the stanniocalcin isolatedfrom Oncorhynchus kisutch (lower line, see SEQ ID NO:10). The middleline polypeptide FIG. 2 (SEQ ID NO:9) shows the amino acids which thepolypeptide according to the invention and the polypeptide of SEQ IDNO:10 have in common. Such common amino acid residues are not acontiguous sequence, but for ease of listing are provided in SEQ ID NO:9as contiguous sequence. No apparent sequence homology exists beyondamino acid 204 of the stanniocalcin from Oncorhynchus kisutch. Totallength of stanniocalcin isolated from Oncorhynchus kisutch is 256 aminoacids.

FIG. 3 shows the banding pattern of the human stanniocalcin polypeptidefollowing bacterial expression and purification.

FIG. 4 illustrates that the polypeptide of the present invention (HumanSCT) inhibits calcium transport in fish as compared to salmonstanniocalcin (salmon SCT) and a control wherein no stanniocalcin wasinjected.

FIG. 5 is an illustration of the results of injecting rats with apolypeptide of the present invention which shows that renal excretion ofphosphate was significantly reduced compared to the control animals.

In accordance with one aspect of the present invention, there areprovided isolated nucleic acids which encode for the mature polypeptidehaving the deduced amino acid sequence of FIG. 1 (SEQ ID No. 2), or forthe mature polypeptide encoded by the cDNA of the clone deposited asATCC Deposit No. 75652 on Jan. 25, 1994.

The ATCC number referred to above is directed to a biological depositwith the ATCC, 12301 Parklawn Drive, Rocville, Md. 20852. Since thestrain referred to is being maintained under the terms of the BudapestTreaty, it will be made available to a patent office signatory to theBudapest Treaty.

The polynucleotide of the present invention was isolated from a humanearly stage lung cDNA library. It contains an open reading frameencoding a prepropolypeptide of 247 amino acids, wherein the first 33amino acids represent a putative leader sequence such that the matureactive polypeptide comprises 214 amino acids. The polypeptide exhibits ahigh degree of homology to Stanniocalcin from Anguilla australis, with119 identical amino acids (61%) in a 195 amino acid overlap. It also hasa very high homology to stanniocalcin from Oncorhynchus kisutch, with118 identical amino acids (57%) in a 204 amino acid overlap.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID No. 1) or that of thedeposited clone or may be a different coding sequence, which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same, mature polypeptide as the DNA of FIG. 1 or thedeposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG. 1(SEQ ID No. 2) or for the mature polypeptide encoded by the depositedcDNA may include: only the coding sequence for the mature polypeptide;the coding sequence for the mature polypeptide and additional codingsequence such as a leader or secretory sequence or a proproteinsequence; the coding sequence for the mature polypeptide (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5' and/or 3' of the coding sequence for the maturepolypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID No. 2) or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 (SEQ ID No. 2) or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 (SEQ ID No. 2) or thepolypeptide encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID No. 1) or of the coding sequence of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5' amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains. Thus,for example, the polynucleotide of the present invention may encode fora mature protein, or for a protein having a prosequence or for a proteinhaving both a prosequence and a presequence (leader sequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. Preferably,the marker sequence is a hexa-histidine tag supplied by a pQE-9 vector(Qiagen, Inc., Chatsworth, Calif.) to provide for purification of themature polypeptide fused to the marker in the case of a bacterial host,or, for example, the marker sequence may be a hemagglutinin (HA) tagwhen a mammalian host, e.g. COS-7 cells, is used. The HA tag correspondsto an epitope derived from the influenza hemagglutinin protein (Wilson,I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term "stringent conditions" means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of FIG. 1 (SEQ ID No. 1) orthe deposited cDNA.

Alternatively, the polynucleotide may be a polynucleotide which has atleast 20 bases, preferably 30 bases, and more preferably at least 50bases which hybridize to a polynucleotide of the present invention andwhich has an identity thereto, as hereinabove described, and which doesnot retain activity. Such polynucleotides may be employed as probes forthe polynucleotide of SEQ ID No. 1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

The deposit(s) referred to herein will be maintained under the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the purposes of Patent Procedure. These deposits are provided merelyas a convenience and are not an admission that a deposit is requiredunder 35 U.S.C. β 112. The sequence of the polynucleotides contained inthe deposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with the description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

The present invention further relates to a Corpuscles of Stanniuspolypeptide which has the deduced amino acid sequence of FIG. 1 (SEQ IDNo. 2) or which has the amino acid sequence encoded by the depositedcDNA, as well as fragments, analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIG. 1 (SEQ ID No. 2) or that encoded by the depositedcDNA, means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNo. 2) or that encoded by the deposited cDNA may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide, for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the Human Corpuscles of Stannius genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their ##STR1## Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain a gene to providea phenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as HEK, CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBs, pD10, phagescript, PsiX174, Pbluescript SK, Pbsks, pNH8a, pNH16a,pNH18a, pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: PWLNEO, pSV2cat, pOG44, pXT1, PSG (Stratagene)pSVK3, PBPV, PMSG, PSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P , P andtrp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptide of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), -factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

Corpuscles of Stannius protein is recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxyapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated with mammalian or other eukaryotic carbohydrates or may benon-glycosylated. Polypeptides of the invention may also include aninitial methionine amino acid residue.

The polypeptide of the present invention may be employed for therapeutictreatment of numerous electrolyte-based diseases. One cause of arterialhypertension is abnormal Na⁺ transport across the cell wall of thevascular smooth cells due to a defect in or inhibition of the Na--Kpump, another is increased permeability to Na⁺ as has been described insome forms of human hypertension. The net result is increases inintracellular Na⁺, which makes the cell more sensitive tovasoconstrictive agents. Since Ca⁺⁺ follows Na⁺, it is postulated thatit is the accumulation of intracellular Ca⁺⁺ and not Na⁺ per se that isresponsible for increased sensitivity to sympathetic stimulation.

The results of Example 4, as shown in FIG. 4, suggest that humanCorpuscles of Stannius protein is an effective inhibitor of calciumuptake when compared to the amount of calcium uptake in its absence(control).

Accordingly, since Corpuscles of Stannius protein can function as ahypocalcemic agent it can help to offset this increased intracellularCa⁺⁺ and reduce or prevent hypertension. Further, hypercalcemia has beenimplicated in heart dysrhythmias, coma and cardiac arrest. Accordingly,the Corpuscles of Stannius protein may have therapeutic value for thetreatment of these disorders by lowering the concentration of freecalcium.

Hypertension is also directly related to renal disorders. Accordingly, ahigher or lower than normal concentration of electrolytes can causerenal malfunction and directly lead to other more serious disorders. Asan example calcium-phosphorus imbalance can cause muscle and bone pain,demineralization of the bones and calcification in various organsincluding the brain, eyes, myocardium and blood vessels.

As shown in Example 5, the results of which are depicted in FIG. 5, theCorpuscles of Stannius protein of the present invention regulates renalexcretion of phosphate. Accordingly, the polypeptide of the presentinvention may be employed to offset disorders that are due to acalcium-phosphate imbalance. Renal failure itself leads to an abnormallyhigh concentration of phosphate in the blood which can be reduced tonormal concentrations by the polypeptide of the present invention.

Similarly, the polypeptide of the present invention may also be employedfor the treatment of certain bone diseases. For example, excessiveconcentrations of calcium lead to the development of fibrous nodules inaffected bone.

The causes of hypercalcemia may also be a number of different disordersincluding hyperparathyroidism, hypervitaminosis D, tumors that raise theserum calcium levels by destroying bone, sarcoidosis, hyperthyroidism,adrenal insufficiency, loss of serum albumin secondary to renaldiseases, excessive GI calcium absorption and elevated concentration ofplasma proteins. Accordingly, Corpuscles of Stannius protein iseffective in reducing hypercalcemia and its related disorders.

Corpuscles of Stannius protein may also be employed for the treatment ofother disorders relating to unusual electrolyte concentrations and fluidimbalance, for example, migraine headaches.

The polynucleotides and polypeptides encoded by such polynucleotides mayalso be utilized for in vitro purposes related to scientific research,synthesis of DNA and manufacture of DNA vectors and for designingtherapeutics and diagnostics for the treatment of human disease.

Fragments of the full length Corpuscles of Stannius gene may be used asa hybridization probe for a cDNA library to isolate the full length geneand to isolate other genes which have a high sequence similarity to theCorpuscles of Stannius gene or similar biological activity. Probes ofthis type generally have at least 20 bases. Preferably, however, theprobes have at least 30 bases and may contain, for example, 50 or morebases. In many cases, the probe has from 20 to 50 bases. The probe mayalso be used to identify a cDNA clone corresponding to a full lengthtranscript and a genomic clone or clones that contain the completeCorpuscles of Stannius gene including regulatory and promotor regions,exons, and introns. An example of a screen comprises isolating thecoding region of the Corpuscles of Stannius gene by using the known DNAsequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of human cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

This invention is also related to the use of the Corpuscles of Stanniusgene as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutations in theCorpuscles of Stannius nucleic acid sequences. Such diseases are relatedto under-expression of the Corpuscles of Stannius polypeptides, forexample, elevated calcium concentration.

Individuals carrying mutations in the Corpuscles of Stannius gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding Corpuscles ofStannius protein can be used to identify and analyze Corpuscles ofStannius gene mutations. For example, deletions and insertions can bedetected by a change in size of the amplified product in comparison tothe normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled Corpuscles of Stannius RNA oralternatively, radiolabeled Corpuscles of Stannius antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of Corpuscles of Stannius protein in various tissuessince an over-expression of the proteins compared to normal controltissue samples may detect the presence of a disease or susceptibility toa disease. Assays used to detect levels of Corpuscles of Stanniusprotein in a sample derived from a host are well-known to those of skillin the art and include radioimmunoassays, competitive-binding assays,Western Blot analysis, ELISA assays and "sandwich" assay. An ELISA assay(Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6,(1991)) initially comprises preparing an antibody specific to theCorpuscles of Stannius antigen, preferably a monoclonal antibody. Inaddition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or, in this example, a horseradishperoxidase enzyme. A sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein, for example, bovine serumalbumen (BSA). Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any Corpuscles ofStannius proteins attached to the polystyrene dish. All unboundmonoclonal antibody is washed out with buffer. The reporter antibodylinked to horseradish peroxidase is now placed in the dish resulting inbinding of the reporter antibody to any monoclonal antibody bound toCorpuscles of Stannius. Unattached reporter antibody is then washed out.Peroxidase substrates are then added to the dish and the amount of colordeveloped in a given time period is a measurement of the amount ofCorpuscles of Stannius protein present in a given volume of patientsample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific toCorpuscles of Stannius protein are attached to a solid support andlabeled Corpuscles of Stannius protein and a sample derived from thehost are passed over the solid support and the amount of label detected,for example by liquid scintillation chromatography, can be correlated toa quantity of Corpuscles of Stannius protein in the sample.

A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assayCorpuscles of Stannius is passed over a solid support and binds toantibody attached to a solid support. A second antibody is then bound tothe Corpuscles of Stannius protein. A third antibody which is labeledand specific to the second antibody is then passed over the solidsupport and binds to the second antibody and an amount can then bequantified.

This invention provides a method for identification of the receptors forthe Corpuscles of Stannius polypeptides. The gene encoding the receptorcan be identified by numerous methods known to those of skill in theart, for example, ligand panning and FACS sorting (Coligan, et al.,Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, and a cDNA library createdfrom this RNA is divided into pools and used to transfect COS cells orother cells that are not responsive to the polypeptides. Transfectedcells which are grown on glass slides are exposed to the labeledpolypeptides. The polypeptides can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to autoradiographic analysis. Positive pools areidentified and sub-pools are prepared and re-transfected using aniterative sub-pooling and re-screening process, eventually yielding asingle clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

This invention provides a method of screening compounds to identifyagonists and antagonists to the human Corpuscles of Stanniuspolypeptides of the present invention. An agonist is a compound whichhas similar biological functions of the polypeptides, while antagonistsblock such functions. An example of such a bioassay comprises injectinghuman Corpuscles of Stannius protein into a species of fish and exposingthe fish to labeled calcium in the presence of the compound to bescreened. A comparative control assay is also performed. The ability ofthe compound to inhibit uptake of calcium, or increase the uptake, canthen be quantified such as by liquid scintillation spectrophotometry.

Alternatively, a mammalian cell or membrane preparation expressing thereceptors of the polypeptide would be incubated with a labeled humanCorpuscles of Stannius polypeptide, eg. by radioactivity, in thepresence of the compound. The ability of the compound to block orenhance this interaction could then be measured.

Examples of potential Corpuscles of Stannius antagonists includeantibodies, or in some cases, oligonucleotides, which bind to thepolypeptides. Another example of a potential antagonist is a negativedominant mutant of the polypeptide. Negative dominant mutants arepolypeptides which bind to the receptor of the wild-type polypeptide,but fail to retain biological activity.

Antisense constructs prepared using antisense technology are alsopotential antagonists. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.For example, the 5' coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (Triple- helix, seeLee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science,241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), therebypreventing transcription and the production of the human Corpuscles ofStannius polypeptide. The antisense RNA oligonucleotide hybridizes tothe mRNA in vivo and blocks translation of the mRNA molecule into thepolypeptides (Antisense--Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). The oligonucleotides described abovecan also be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of the human Corpuscles ofStannius polypeptides.

Another potential human Corpuscles of Stannius antagonist is a peptidederivative of the polypeptides which are naturally or syntheticallymodified analogs of the polypeptides that have lost biological functionyet still recognize and bind to the receptors of the polypeptides tothereby effectively block the receptors. Examples of peptide derivativesinclude, but are not limited to, small peptides or peptide-likemolecules.

The antagonists may be employed to inhibit the hypocalcemic effects ofthe human Corpuscles of Stannius protein, and to treat osteoporosis andPaget's Diseases, among other disorders where an increase of calciumlevels is desired.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The human Corpuscles of Stannius protein and agonists and antagonistsmay be employed in combination with a suitable pharmaceutical carrier.Such compositions comprise a therapeutically effective amount of thepolypeptide, antagonist or agonist and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides, agonists or antagonists may be employed in conjunctionwith other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the topical, intravenous, intraperitoneal,intramuscular, intratumor, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, the polypeptides will be administered in anamount of at least about 10 g/kg body weight and in most cases they willbe administered in an amount not in excess of about 8 mg/Kg body weightper day. In most cases, the dosage is from about 10 g/kg to about 1mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc.

The human Corpuscles of Stannius protein, and agonists or antagonistswhich are polypeptides, may be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding the polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding the polypeptide of the presentinvention.

Similarly, cells may be engineered in vivo for expression of thepolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

The retroviral plasmid vectors may be derived from retroviruses whichinclude, but are not limited to, Moloney Murine Sarcoma Virus, MoloneyMurine Leukemia Virus, spleen necrosis virus, Rous Sarcoma Virus andHarvey Sarcoma Virus.

In a preferred embodiment the retroviral expression vector, pMV-7, isflanked by the long terminal repeats (LTRs) of the Moloney murinesarcoma virus and contains the selectable drug resistance gene neo underthe regulation of the herpes simplex virus (HSV) thymidine kinase (tk)promoter. Univque EcoRI and HimdIII sites facilitate the introduction ofcoding sequence (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)).

The vectors include one or more suitable promoters which include, butare not limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller, et al.,Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter(e.g., cellular promoters such as eukaryotic cellular promotersincluding, but not limited to, the histone, pol III, and -actinpromoters). The selection of a suitable promoter will be apparent tothsoe skilled in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter which includes,but is not limited to, viral thymidine kinase promoters, such as theHerpes Simplex thymidine kinase promoter; retroviral LTRs, the -actinpromoter, and the native promoter which controls the gene encoding thepolypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317 andGP+am12. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO precipitation.

The producer cell line generates infectious ##STR2## vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced, include but arenot limited to, fibroblasts and endothelial cells.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3'untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 500 or 600 bases; however, clones larger than that have ahigher likelihood of binding to a unique chromosomal location withsufficient signal intensity for simple detection. For example, 2,000 bpis good, 4,000 is better, and more than 4,000 is probably not necessaryto get good results a reasonable percentage of the time. For a review ofthis technique, see Verma et al., Human Chromosomes: a Manual of BasicTechniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1

Bacterial Expression and Purification of Corpuscles of Stannius Protein

The DNA sequence encoding Corpuscles of Stannius Protein, ATCC # 75652,is initially amplified using PCR oligonucleotide primers correspondingto the 5' and 3' end sequences of Corpuscles of Stannius nucleic acidsequence. Additional nucleotides corresponding to the SphI and BglIIrestriction enzyme site were added to the 5' and 3' sequencesrespectively. The 5' oligonucleotide primer has the sequence 5'GACTGCATGCTCCAAAACTCAGCAGTG 3' (SEQ ID No. 3), contains a SphIrestriction enzyme site and 21 nucleotides of Corpuscles of StanniusProtein coding sequence starting from the initiation codon; the 3'sequence 3' GACTAGATCTTGCACTCTCATGGGATGTGCG 5' (SEQ ID No. 4) containscomplementary sequences to a BglII restriction site (AGATCT) and thelast 21 nucleotides of Corpuscles of Stannius Protein coding sequence.The restriction enzyme sites correspond to the restriction enzyme siteson the bacterial expression vector pQE70 (Qiagen, Inc. Chatsworth,Calif.). pQE70 encodes antibiotic resistance (Amp^(r)), a bacterialorigin of replication (ori), an IPTG-regulatable promoter operator(P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzymesites. pQE70 was then digested with the SphI and BglII restrictionenzymes. The amplified sequences were ligated into pQE70 and wereinserted in frame with the sequence encoding for the histidine tag andthe RBS. The ligation mixture was then used to transform E. coli strainM15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmidpREP4, which expresses the lacI repressor and also confers kanamycinresistance (Kan^(r)). Transformants are identified by their ability togrow on LB plates and ampicillin/kanamycin resistant colonies wereselected. Plasmid DNA was isolated and confirmed by restrictionanalysis. Clones containing the desired constructs were grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). Tho O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalacto pyranoside) was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 3 to 4 hours. Cells were then harvested by centrifugation (20mins at 6000×g). The cell pellet was solubilized in the chaotropic agent6 Molar Guanidine HCl. After clarification, solubilized stanniocalcinwas purified from this solution by chromatography on a Nickel-Chelatecolumn under conditions that allow for tight binding by proteinscontaining the 6-His tag (Hochuli, E. et al., Genetic Engineering,Principles & Methods, 12:87-98 (1990). Protein renaturation out of GnHClcan be accomplished by several protocols (Jaenicke, R. and Rudolph, R.,Protein Structure--A Practical Approach, IRL Press, New York (1990)).Initially, step dialysis is utilized to remove the GnHCL. Alternatively,the purified protein isolated from the Ni-chelate column can be bound toa second column over which a decreasing linear GnHCL gradient is run.The protein is allowed to renature while bound to the column and issubsequently eluted with a buffer containing 250 mM Imidazole, 150 mMNaCl, 25 mM Tris-HCl pH 7.5 and 10% Glycerol. Finally, soluble proteinis dialyzed against a storage buffer containing 5 mM AmmoniumBicarbonate. The purified protein was analyzed by SDS-PAGE (FIG. 3).

EXAMPLE 2

Cloning and Expression of Human Stanniocalcin Using the BaculovirusExpression System

The DNA sequence encoding the full length human Stanniocalcin protein,ATCC # 75652, was amplified using PCR oligonucleotide primerscorresponding to the 5' and 3' sequences of the gene:

The 5' primer has the sequence 5' CAGTGGATCCGCCACCATGCTCCAAAACTCAGCAGTG3' (SEQ ID No. 5) and contains a BamHI restriction enzyme site (in bold)followed by 6 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196:947-950 (1987) which is just behind the first 21 nucleotides of thehuman Corpuscles of Stannius gene (the initiation codon for translation"ATG" is underlined).

The 3' primer has the sequence 5' CAGTGGTACCGGTTGTGAATAACCTCTCCC 3' (SEQID No. 6) and contains the cleavage site for the restrictionendonuclease Asp718 and 20 nucleotides complementary to the 3'non-translated sequence of the Corpuscles of Stannius gene. The fragmentwas digested with the endonucleases BamHI and Asp718 and then purifiedagain on a 1% agarose gel. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the human Corpuscles of Stannius protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby the recognition sites for the restriction endonucleases BamHI andAsp718. The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid was digested with the restriction enzymes BamHI and Asp718and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA was then isolated from a 1% agarosegel using the commercially available kit ("Geneclean" BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNAligase. E. coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBac-hSTC) with the humanCorpuscles of Stannius gene using the enzymes BamHI and Asp718. Thesequence of the cloned fragment was confirmed by DNA sequencing.

5 μg of the plasmid pBac-hSTC was co-transfected with 1.0 μg of acommercially available linearized baculovirus ("BaculoGold baculovirusDNA", Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold virus DNA and 5 μg of the plasmid pBac-hSTC weremixed in a sterile well of a microtiter plate containing 50 μl of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 278° C. After 5hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at278° C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with "Blue Gal" (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a "plaque assay" can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the virus was added to the cellsand blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculovirus was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 48° C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-hSTC at a multiplicity of infection (MOI) of 2. Six hourslater the medium was removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵ S-methionine and 5 μCi ³⁵ S cysteine (Amersham) wereadded. The cells were further incubated for 16 hours before they wereharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 3

Cloning and Expression of Human Corpuscles of Stannius Protein UsingChinese Hamster Ovary Cells Lacking Dihydrofolate Activity

The vector pN346 is used for the expression of the human Corpuscles ofStannius protein. Plasmid pN346 is a derivative of the plasmid pSV2-DHFR[ATCC Accession No. 37146]. Both plasmids contain the mousedihydrolfolate reductase (DHFR) gene under control of the SV40 earlypromoter. Chinese hamster ovary, or other cells, lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate(MTX). The amplification of the DHFR genes in cells resistant tomethotrexate has been well documented (see, e.g., Alt, F. W., Kellems,R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol.9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

Plasmid pN346 contains a strong promoter for the expression of the geneof interest, namely, the long terminal repeat (LTR) of the Rous SarcomaVirus (Cullen et al., Molecular and Cellular Biology, March 1985,438-447) plus a fragment isolated from the enhancer of the immediateearly gene of human cytomegalovirus (CMV) (Boshart et al., Cell41:521-530, 1985). Downstream of the promoter are the following singlerestriction enzyme cleavage sites that allow the integration of thegenes; BamHI, PvuII, and NruI. Behind these cloning sites, the plasmidcontains translational stop codons in all three reading frames followedby the 3' intron and the polyadenylation site of the rat preproinsulingene. Other high efficient promoters can also be used for expression,e.g., the human -actin promoter, the SV40 early or late promoters or thelong terminal repeats from other retroviruses, e.g., HIV and HTLVI. Forthe polyadenylation of mRNA, other signals, e.g., from the human growthhormone or globin genes, may be used as well.

Stable cell lines carrying a gene of interest integrated into thechromosome can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pN346 was digested with the restriction enzyme BamHI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The vector was then isolated from a 1% agarose gel.

The DNA sequence encoding human Corpuscles of Stannius protein, ATCC #75652, was amplified using PCR oligonucleotide primers corresponding tothe 5' and 3' sequences of the gene:

The 5' primer has the sequence 5' CAGTGGATCCGCCACCATGCTCCAAAACTCAGCAGTG3' (SEQ ID No. 7) and contains a BamHI restriction enzyme site (in bold)followed by 6 nucleotides resembling the efficient signal fortranslation (Kozak, M., supra) plus the first 21 nucleotides of the gene(the initiation codon for translation "ATG" is underlined).

The 3' primer has the sequence 5' CAGTGGATCCGGTTGTGAATAACCTCTCCC 3' (SEQID No. 8) and contains the cleavage site for the restrictionendonuclease BamHI and 20 nucleotides complementary to the 3'non-translated sequence of the gene.

The amplified fragments digested with the endonuclease BamHI and thenpurified on a 1% agarose gel.

The isolated fragment and the dephosphorylated vector were then ligatedwith T4 DNA ligase. E. coli HB101 cells were then transformed andbacteria identified that contained the plasmid pN346hSTC inserted in thecorrect orientation. The sequence of the inserted gene was confirmed byDNA sequencing.

Transfection of CHO-DHFR-Cells

Chinese hamster ovary cells lacking an active DHFR enzyme were used fortransfection. 5 μg of the expression plasmid pN346hSTC wereco-transfected with 0.5 μg of the plasmid pSVneo using the lipofectionmethod (Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the gene neo from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells wereseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells were trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) and cultivated for 10-14 days. After this period,single clones were trypsinized and then seeded in 6-well petri dishesusing different concentrations of methotrexate (25, 50 nm, 100 nm, 200nm, 400 nm). Clones growing at the highest concentrations ofmethotrexate were then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). Thesame procedure was repeated until clones grew at a concentration of 100μM.

The expression of the desired gene product was analyzed by Western blotanalysis and SDS-PAGE.

EXAMPLE 4

Human Corpuscles of Stannius Protein Inhibits Calcium Uptake in Fish

Goldfish (1.2-0.1 g; 10 fish per group were given intraperitonealinjections of bacterial expressed human STC (10 mg/kg), salmon STC (1mg/kg) or saline and placed in tanks of ⁴⁵ Ca water (50.000 dpm/ml) for3 hours. The fish were sacrificed, ashed overnight at 6008° C. and theisotope content of the ash was determined by scintillation counting.Based on body weight and the specific activity of the water, whole bodyCa⁺⁺ uptake in each fish was expressed as μM Ca2+kg/hour. Both human andsalmon STC had statistically significant inhibitory effects on gill Ca2+transport at the doses employed (P<0.05, P,0.01) demonstrating that thebacterial expressed human stanniocalcin has biological activity in fish(FIG. 4).

EXAMPLE 5

Bacterial Expressed Human Corpuscles of Stannius Protein Reduces RenalExcretion of Phosphate

Rats (250±10 g; 5 rats per group) were anesthetized with inactin andcatheters were placed in the jugular vein and carotid arteries. Theurethras were also catheterized for collection of urine. The animalswere continuously infused with inulin and PAH, used for small unreactivecompounds for the measurement of glomerular filtration and renal bloodflow, respectively. The animals were also connected to a physiograph formonitoring of blood pressure and heart rate. Blood samples were takenfrom the carotid catheter. Animals were given a bolus injection of humanCorpuscles of Stannius or saline via the jugular catheter after thefirst urine collection period (arrow) and monitored for changes inplasma and urinary electrolytes, renal function, heart rate and bloodpressure.

Shown are the effects of a 5 n moles/kg body weight bolus injection ofhuman stanniocalcin, delivered over 5 minutes (arrow). Controls receivedan equivalent volume of saline. Human stanniocalcin had no effect onheart rate or blood pressure (P<0.05, Students T-test). Excretion ofelectrolytes was measured as absolute excretion or as a proportion ofrenal filtered load reabsorbed by the kidneys (fractional excretion,FE). Only data for absolute excretion of phosphate was demonstrated tobe significantly different from control animals. Plasma levels ofcalcium, phosphate, potassium and sodium did not significantly change.Absolute excretion of calcium and fractional excretion of calcium andphosphate did not change significantly. Absolute phosphate excretion wasmaximally suppressed 60 minutes after hormone delivery and remainedsignificantly lower than the saline-injected controls until the end ofthe experiment (P<0.02) (FIG. 5).

EXAMPLE 6

Purification of Baculovirus-Expressed Human Corpuscles of StanniusProtein

The human Stanniocalcin gene was cloned into the pRG1 baculovirustransfer vector. Culture supernatant from SF9 cells infected withrecombinant baculovirus was collected, concentrated 10 times, anddiafiltered to 50 mM Tris pH 7.0 and 500 mM NaCl. After centrifugation,the solution was applied to a conA column and washed using the samebuffer followed by washing with 50 mM tris pH 7.0 and 50 mM NaCl.Following elution in 50 mM Tris, 100 mM Borate, 750 mM mannose and 750mM dextrose, an equal volume of 50 mM MES pH 5.8 was added and thesolution was applied to an ion exchange column (S column) and proteineluted by a gradient of NaCl.

Shown are four fractions eluted from the S column at 200-300 mM NaCl(FIG. 6). A) Analysis by SDS-PAGE, and B) Western blotting using arabbit antibody raised against fish Stanniocalcin.

EXAMPLE 7

N-terminal Sequencing of Baculovirus Expressed Human Corpuscles ofStannius

The material for sequencing Run #19 was obtained frombaculovirus-expressed Corpuscles of Stannius by first applying thesupernatant to a Con A column equilibrated with 50 mM Tris, pH 7.0,containing 0.5 M NaCl, 1 mM CaCl , 1 mM MgC1 and 1 mM MgC1 . TheCorpuscles of Stannius was eluted with the above buffer containing 100mM sodium ##STR3## at pH 7.0, 0.75 M ##STR4## and 0.75 M mannose. Thisfraction was then further purified using a reversed phase HPLC column(RP-300, 2.1×30 mm) equilibrated with 0.1% TFA (Solvent A). The proteinswere eluted with a 7.5 min. gradient from 0% to 60% Solvent B(acetonitrile containing 0.07% TFA). A fraction which was positive byWestern blot analysis was then sequenced (Run #19). The material forsequencing Run #26 was obtained from a supernatant containingbaculovirus-expressed Corpuscles of Stannius which was concentrated10-fold and diafiltered with 50 mM TrisHCl at pH 7.0 containing 500 mMNaCl. This concentrate was then applied to a Con A column equilibratedwith this pH 7.0 Tris buffer, washed with 50 mM Tris-HCl at pH 7.0containing 20 mM NaCl and then eluted using the same 50 mM Tris-HCl atpH 7.0 containing 20 mM NaCl with 100 mM sodium borate at pH 7.0, 0.75 Mmannose and 0.75M dextrose. The fractions were pooled and an equalvolume of 50 mM MES buffer at pH 5.8 was added. This solution was thenapplied to a SP-650M column (1.0×6.6 cm, Toyopearl) at a flow rate of 1ml/min. Proteins were then eluted with step gradients at 200, 300 and500 mM NaCl. The Corpuscles of Stannius was obtained using the elutionat 300 mM NaCl. The partially purified material was spotted onto theProBlott membrane (Applied Biosystems, Inc.) and subjected to amino acidsequence analysis. The underlined letters represent the N-terminalresidues as determined by amino acid sequence analysis, and are reportedas the N-terminal amino acid sequence of SEQ ID NO:2. The lower caseletters are residues that could not be conclusively identified, but theresults are consistent with the expected sequence. The colons indicatewhere the observed residues are identical to that expected from DNAsequencing.

    __________________________________________________________________________                         (Run#26)                                                                    THEAEONDSVSPrK (SEQ ID NO:2, amino acids -18 to -5)                          .linevert split..linevert split..linevert split..linever    t split..linevert split..linevert split..linevert split..linevert             split..linevert split..linevert split..linevert split..linevert split..lin    evert split..linevert split.                                                   MLQNSAVLLVLVISASATHEAEQNDSVSPRKSRVA                                                               .linevert split..linevert split..linevert split..line    vert split..linevert split..linevert split..linevert split..linevert          split..linevert split..linevert split..linevert split..linevert split..lin    evert split..linevert split..linevert split.                                                       AEONDSVSPRKsrva (SEQ ID NO:2, amino acids -15 to -1)                             (Run #26)                                               -                      (Run#19)                                                               AONSAEVVRcLNSAL (SEQ ID NO:2, amino acids 1 to 15)                            .linevert split..linevert split..linevert split..linever    t split..linevert split..linevert split..linevert split..linevert             split..linevert split..linevert split..linevert split..linevert split..lin    evert split..linevert split..linevert split.                                                    AQNSAEVVRCLNSALQVGCGAFACLE                                    - NSTCDTDGMYDICKSFLYSAAKFDTQGKAFVKESLKCIANGVTSKVRLAIRRCSTFQRMIAEVQEECYSK    LN                                                                             VCSIAKRNPEAITEVVQLPNHFSNRYYNRLVRSLLECDEDTVSTIRDSLMEKIGPNMASLFHILQTDHCAQT      HPRADFNRRRTNEPQKLKVLLRNLRGEEDSPSHIKRTSHESA                                   __________________________________________________________________________

As shown above, the N-terminal signal sequence begins with the initialmethianine residue and ends between the two alanine residues at position35 and 36. This has been deduced since a full-length protein is firstprocessed between the alanine and threonine residues at positions 17 and18 and is then further processed between the two alanine residues atpositions 34 and 35 to produce the mature protein.

EXAMPLE 8

Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 378° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5' and 3' end sequencesrespectively. The 5' primer containing an EcoRI site and the 3' primerhaving contains a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the EcoRI and HimdIII fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is used to transform bacteria HB101,which are then plated onto agar-containing kanamycin for the purpose ofconfirming that the vector had the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  8                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  771 BAS - #E PAIRS                                               (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  cDNA                                             - -  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:                     - - GAAACTTCTC AGAGAATGCT CCAAAACTCA GCAGTGCTTC TGGTGCTGGT GA -             #TCAGTGCT     60                                                                 - - TCTGCAACCC ATGAGGCGGA GCAGAATGAC TCTGTGAGCC CCAGGAAATC CC -            #GAGTGGCG    120                                                                 - - GCCCAAAACT CAGCTGAAGT GGTTCGTTGC CTCAACAGTG CTCTACAGGT CG -            #GCTGCGGG    180                                                                 - - GCTTTTGCAT GCCTGGAAAA CTCCACCTGT GACACAGATG GGATGTATGA CA -            #TCTGTAAA    240                                                                 - - TCCTTCTTGT ACAGCGCTGC TAAATTTGAC ACTCAGGGAA AAGCATTCGT CA -            #AAGAGAGC    300                                                                 - - TTAAAATGCA TCGCCAACGG GGTCACCTCC AAGGTCTTCC TCGCCATTCG GA -            #GGTGCTCC    360                                                                 - - ACTTTCCAAA GGATGATTGC TGAGGTGCAG GAAGAGTGCT ACAGCAAGCT GA -            #ATGTGTGC    420                                                                 - - AGCATCGCCA AGCGGAACCC TGAAGCCATC ACTGAGGTCG TCCAGCTGCC CA -            #ATCACTTC    480                                                                 - - TCCAACAGAT ACTATAACAG ACTTGTCCGA AGCCTGCTGG AATGTGATGA AG -            #ACACAGTC    540                                                                 - - AGCACAATCA GAGACAGCCT GATGGAGAAA ATTGGGCCTA ACATGGCCAG CC -            #TCTTCCAC    600                                                                 - - ATCCTGCAGA CAGACCACTG TGCCCAAACA CACCCACGAG CTGACTTCAA CA -            #GGAGACGC    660                                                                 - - ACCAATGAGC CGCAGAAGCT GAAAGTCCTC CTCAGGAACC TCCGAGGTGA GG -            #AGGACTCT    720                                                                 - - CCCTCCCACA TCAAACGCAC ATCCCATGAG AGTGCATAAC CAGGGAGAGG T - #                771                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  247 AMI - #NO ACIDS                                              (B) TYPE:  AMINO ACI - #D                                                     (C) STRANDEDNESS:                                                             (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  PROTEIN                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:                          - - Met Leu Gln Asn Ser Ala Val Leu Leu Val Le - #u Val Ile Ser Ala         35                 - - #30                 - - #25                              - - Ser Ala Thr His Glu Ala Glu Gln Asn Asp Se - #r Val Ser Pro Arg         20                 - - #15                 - - #10                              - - Lys Ser Arg Val Ala Ala Gln Asn Ser Ala Gl - #u Val Val Arg Cys         5                  - #  1               5 - #                 10                - - Leu Asn Ser Ala Leu Gln Val Gly Cys Gly Al - #a Phe Ala Cys Leu                          15  - #                20  - #                25               - - Glu Asn Ser Thr Cys Asp Thr Asp Gly Met Ty - #r Asp Ile Cys Lys                          30  - #                35  - #                40               - - Ser Phe Leu Tyr Ser Ala Ala Lys Phe Asp Th - #r Gln Gly Lys Ala                          45  - #                50  - #                55               - - Phe Val Lys Glu Ser Leu Lys Cys Ile Ala As - #n Gly Val Thr Ser                          60  - #                65  - #                70               - - Lys Val Phe Leu Ala Ile Arg Arg Cys Ser Th - #r Phe Gln Arg Met                          75  - #                80  - #                85               - - Ile Ala Glu Val Gln Glu Glu Cys Tyr Ser Ly - #s Leu Asn Val Cys                          90  - #                95  - #               100               - - Ser Ile Ala Lys Arg Asn Pro Glu Ala Ile Th - #r Glu Val Val Gln                          105  - #               110  - #               115              - - Leu Pro Asn His Phe Ser Asn Arg Tyr Tyr As - #n Arg Leu Val Arg                          120  - #               125  - #               130              - - Ser Leu Leu Glu Cys Asp Glu Asp Thr Val Se - #r Thr Ile Arg Asp                          135  - #               140  - #               145              - - Ser Leu Met Glu Lys Ile Gly Pro Asn Met Al - #a Ser Leu Phe His                          150  - #               155  - #               160              - - Ile Leu Gln Thr Asp His Cys Ala Gln Thr Hi - #s Pro Arg Ala Asp                          165  - #               170  - #               175              - - Phe Asn Arg Arg Arg Thr Asn Glu Pro Gln Ly - #s Leu Lys Val Leu                          180  - #               185  - #               190              - - Leu Arg Asn Leu Arg Gly Glu Glu Asp Ser Pr - #o Ser His Ile Lys                          195  - #               200  - #               205              - - Arg Thr Ser His Glu Ser Ala                                                              210                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  27 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:                          - - GACTGCATGC TCCAAAACTC AGCAGTG          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  31 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #4:                          - - GACTAGATCT TGCACTCTCA TGGGATGTGC G        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  37 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #5:                          - - CAGTGGATCC GCCACCATGC TCCAAAACTC AGCAGTG      - #                       - #      37                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  30 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #6:                          - - CAGTGGTACC GGTTGTGAAT AACCTCTCCC         - #                  - #               30                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  37 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #7:                          - - CAGTGGATCC GCCACCATGC TCCAAAACTC AGCAGTG      - #                       - #      37                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  30 BASE - # PAIRS                                                (B) TYPE:  NUCLEIC A - #CID                                                   (C) STRANDEDNESS:  SING - #LE                                                 (D) TOPOLOGY:  LINEAR                                                - -     (ii) MOLECULE TYPE:  Oligonucleotide                                  - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #8:                          - - CAGTGGATCC GGTTGTGAAT AACCTCTCCC         - #                  - #               30                                                                   __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide comprising a memberselected from the group consisting of:(a) a polypeptide having an aminoacid sequence according to amino acids 1 to 212 of SEQ ID NO:2; (b) apolypeptide which is a fragment of the amino acid sequence according toSEQ ID NO:2, wherein said fragment retains the calcium uptake inhibitoryactivity of the polypeptide according to SEQ ID NO:2; and (c) apolypeptide consisting of amino acid residues 1 to 20 of SEQ ID NO:2. 2.A polypeptide according to claim 1, comprising a member selected from(a), (b) or (c) and further comprising a pro-sequence that may becleaved to provide a polypeptide comprising an active (a), (b) or (c)polypeptide.
 3. An isolated polypeptide according to claim 1, comprisingamino acids 1 to 212 of SEQ ID NO:2.
 4. An isolated polypeptideaccording to claim 2, wherein said polypeptide comprises amino acids -35to 212 of SEQ NO:2.
 5. An isolated polypeptide according to claim 1,wherein said member is (b).
 6. An isolated polypeptide comprising amember selected from the group consisting of:(a) a polypeptide encodedby the portion of the human cDNA of ATCC Deposit No. 75652 which encodesthe mature polypeptide; (b) a polypeptide which is a fragment (a),wherein said fragment retains the calcium uptake inhibitory activity ofthe polypeptide of (a); and (c) a polypeptide consisting of theN-terminal 20 amino acids of the polypeptide encoded by the human cDNAof ATCC Deposit No.
 75652. 7. A polypeptide according to claim 6,comprising a member selected from (a), (b) or (c) and further comprisinga pro-sequence that may be cleaved to provide a polypeptide comprisingan active (a), (b) or (c) polypeptide.
 8. An isolated polypeptideaccording to claim 6 comprising the mature polypeptide of member (a). 9.An isolated polypeptide according to claim 7, wherein said polypeptidesequence comprises the polypeptide encoded by the human cDNA of ATCCDeposit No.
 75652. 10. An isolated polypeptide according to claim 6,wherein said member is (b).
 11. A method for the treatment of a patienthaving need to inhibit uptake of calcium comprising administering to thepatient a therapeutically effective amount of the polypeptide ofclaim
 1. 12. A method for the treatment of a patient having need toinhibit uptake of calcium comprising administering to the patient atherapeutically effective amount of the polypeptide of claim
 3. 13. Amethod for the treatment of a patient having need to inhibit uptake ofcalcium comprising administering to the patient a therapeuticallyeffective amount of the polypeptide of claim
 5. 14. A method for thetreatment of a patient having need to inhibit uptake of calciumcomprising administering to the patient a therapeutically effectiveamount of the polypeptide of claim
 6. 15. A method for the treatment ofa patient having need to inhibit uptake of calcium comprisingadministering to the patient a therapeutically effective amount of thepolypeptide of claim
 8. 16. A method for the treatment of a patienthaving need to inhibit uptake of calcium comprising administering to thepatient a therapeutically effective amount of the polypeptide of claim10.